Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.5.1.4 (deaminase)
5,113 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

An N-acyl-d-amino acid amidohydrolase (N-D-AAase) was identified in cell extracts of a strain, Iso1, isolated from an environment containing N-acetyl-d-methionine. The bacterium was classified as Variovorax paradoxus by phylogenetic analysis. The gene was cloned and sequenced. The gene consisted of a 1467-bp ORF encoding a polypeptide of 488 amino acids. The V. paradoxusN-D-AAase showed significant amino acid similarity to the N-acyl-d-amino acid amidohydrolases of the two eubacteria Alcaligenes xylosoxydans A-6 (44-56% identity), Alcaligenes facelis DA1 (54% identity) and the hyperthermophilic archaeon Pyrococcus abyssi (42% identity). After over-expression of the N-D-AAase protein in Escherichia coli, the enzyme was purified by multistep chromatography. The native molecular mass was 52.8 kDa, which agreed with the predicted molecular mass of 52 798 Da and the enzyme appeared to be a monomer protein by gel-filtration chromatography. A homogenous protein with a specific activity of 516 U.mg-1 was finally obtained. After peptide sequencing by LC/MS/MS, the results were in agreement with the deduced amino acid sequence of the N-D-AAase. The pI of the enzyme was 5.12 and it had an optimal pH and temperature of 7.5 and 50 degrees C, respectively. After 30 min heat treatment at 45 degrees C, between pH 6 and pH 8, 80% activity remained. The N-D-AAase had higher hydrolysing activity against N-acetyl-d-amino acid derivates containing d-methionine, d-leucine and d-alanine and against N-chloroacetyl-d-phenylalanine. Importantly, the enzyme does not act on the N-acetyl-l-amino acid derivatives. The enzyme was inhibited by chelating agents and certain metal ions, but was activated by 1 mm of Co2+ and Mg2+. Thus, the N-D-AAase from V. paradoxus can be considered a chiral specific and metal-dependent enzyme.
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PMID:Identification and characterization of a new gene from Variovorax paradoxus Iso1 encoding N-acyl-D-amino acid amidohydrolase responsible for D-amino acid production. 1235 18

A novel amidase acting on (R,S)-piperazine-2-tert-butylcarboxamide was purified from Pseudomonas sp. MCI3434 and characterized. The enzyme acted R-stereoselectively on (R,S)-piperazine-2-tert-butylcarboxamide to yield (R)-piperazine-2-carboxylic acid, and was tentatively named R-amidase. The N-terminal amino acid sequence of the enzyme showed high sequence identity with that deduced from a gene named PA3598 encoding a hypothetical hydrolase in Pseudomonas aeruginosa PAO1. The gene encoding R-amidase was cloned from the genomic DNA of Pseudomonas sp. MCI3434 and sequenced. Analysis of 1332 bp of the genomic DNA revealed the presence of one open reading frame (ramA) which encodes the R-amidase. This enzyme, RamA, is composed of 274 amino acid residues (molecular mass, 30 128 Da), and the deduced amino acid sequence exhibits homology to a carbon-nitrogen hydrolase protein (PP3846) from Pseudomonas putida strain KT2440 (72.6% identity) and PA3598 protein from P. aeruginosa strain PAO1 (65.6% identity) and may be classified into a new subfamily in the carbon-nitrogen hydrolase family consisting of aliphatic amidase, beta-ureidopropionase, carbamylase, nitrilase, and so on. The amount of R-amidase in the supernatant of the sonicated cell-free extract of an Escherichia coli transformant overexpressing the ramA gene was about 30 000 times higher than that of Pseudomonas sp. MCI3434. The intact cells of the E. coli transformant could be used for the R-stereoselective hydrolysis of racemic piperazine-2-tert-butylcarboxamide. The recombinant enzyme was purified to electrophoretic homogeneity from cell-free extract of the E. coli transformant overexpressing the ramA gene. On gel-filtration chromatography, the enzyme appeared to be a monomer. It had maximal activity at 45 degrees C and pH 8.0, and was completely inactivated in the presence of p-chloromercuribenzoate, N-ethylmaleimide, Mn2+, Co2+, Ni2+, Cu2+, Zn2+, Ag+, Cd2+, Hg2+, or Pb2+. RamA had hydrolyzing activity toward the carboxamide compounds, in which amino or imino group is connected to beta- or gamma-carbon, such as beta-alaninamide, (R)-piperazine-2-carboxamide (R)-piperidine-3-carboxamide, D-glutaminamide and (R)-piperazine-2-tert-butylcarboxamide. The enzyme, however, did not act on the other amide substrates for the aliphatic amidase despite its sequence similarity to RamA.
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PMID:A novel R-stereoselective amidase from Pseudomonas sp. MCI3434 acting on piperazine-2-tert-butylcarboxamide. 1506 83

The chemoenzymatic route to 2-deoxy-2-propionamido-D-mannose (1b), 2-butyramido-2-deoxy-D-mannose (2b) and 2-deoxy-2-phenylacetamido-D-mannose (3b) involved N-acylation of 2-amino-2-deoxy-D-glucose followed by alkaline C-2 epimerization and selective microbial removal of the epimers with gluco-configuration. The latter step employed whole cells of Rhodococcus equi A4 able to degrade 2-deoxy-2-propionamido-D-glucose (1a), 2-butyramido-2-deoxy-D-glucose (2a) and 2-deoxy-2-phenylacetamido-D-glucose (3a) but inactive towards the corresponding manno-isomers. The metabolism of the gluco-isomers probably involved phosphorylation and subsequent deacylation. 2-Acetamido-2-deoxy-6-O-phospho-D-glucose amidohydrolase [EC 3.5.1.25] but not 2-acetamido-2-deoxy-D-glucose amidohydrolase was detected in the cell extract, the former enzyme being partially purified (15.8-fold with an overall yield of 18.1% and a specific activity of 0.95 units mg-1 protein). According to SDS-PAGE electrophoresis, gel filtration and mass spectrometry, the enzyme was a monomer with an apparent molecular mass of approximately 42 kDa. The optimum temperature and pH of the enzyme were 60 degrees C and 8.0-9.0, respectively. 2-Acetamido-2-deoxy-6-O-phospho-D-glucose and 2-acetamido-2-deoxy-6-O-sulfo-D-glucose but not 2-acetamido-2-deoxy-1-O-phospho-D-glucose or 2-acetamido-2-deoxy-D-glucose were substrates of the enzyme. Its activity was slightly inhibited by the addition of 1 mM Al3+, Ca2+, Co2+, Cu2+, Mn2+ or Zn2+ and activated by 1 mM Mg2+. The concentrated enzyme is highly stable at 4 degrees C in the presence of 0.1 M ammonium sulfate.
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PMID:A chemoenzymatic route to mannosamine derivatives bearing different N-acyl groups. 1560 34

The gene cluster containing the nitrile hydratase (NHase) and amidase genes of a moderate thermophile, B. pallidus RAPc8 has been cloned and sequenced. The (5.9 kb) section of cloned DNA contained eight complete open reading frames, encoding (in order), amidase (belonging to the nitrilase related aliphatic amidase family), nitrile hydratase beta and alpha subunits (of the cobalt containing class), a 122-amino acid accessory protein, designated P14K, a homologue of the 2Fe-2S class of ferredoxins and three putative proteins with distinct homology to the cobalt uptake proteins cbiM, cbiN and cbiQ of the S. typhimurium LT2 cobalamin biosynthesis pathway. The amidase and nitrile hydratase genes were subcloned and inducibly expressed in Escherichia coli, to levels of approximately 37 U/mg and 49 U/mg, respectively, without the co-expression of additional flanking genes. However, co-expression of P14K with the NHase structural genes significantly enhanced the specific activity of the recombinant NHase. This is the first description of an accessory protein involved in thermostable NHase expression. Modelling of the P14K protein structure has suggested that this protein functions as a subunit-specific chaperone, aiding in the folding of the NHase alpha subunit prior to alpha-beta subunit association and the formation of alpha(2)beta(2) NHase holoenzyme.
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PMID:Molecular analysis of the nitrile catabolism operon of the thermophile Bacillus pallidus RAPc8. 1595 32

A dicarboxylate monoamide amidohydrolase (half-amidase) was identified from a cyclicimide-metabolizing microorganism, Alcaligenes eutrophus 112R4. The enzyme catalyzed the hydrolysis of monoamidated dicarboxylates, which were the hydrolyzing products of cyclic imides by imidase, to dicarboxylates and ammonia. The enzyme showed high catalytic activity to succinamic acid, but no obvious activity to aliphatic amides, amino acid amides, N-carbamoyl amino acids and urea was observed. The productions of half-amidase and imidase were correlative in Alcaligenes eutrophus 112R4, in that succinimide and succinamic acid enhanced the expressions of these two enzymes simultaneously, while free ammonia repressed their expressions. Succinate showed regulation effects on either synthesis or activities of half-amidase and imidase. The characteristics of half-amidase were investigated by using the crude extract of recombined E. coli cell. The fact that cobalt ion stimulated the activity of half-amidase by a coefficient of 3.37, implied that half-amidase was probably a metal-binding enzyme.
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PMID:[A dicarboxylate monoamide amidohydrolase (half-amidase) from Alcaligenes eutrophus 112R4]. 1627 76

TrzN, the broad-specificity triazine hydrolase from Arthrobacter and Nocardioides spp., is reportedly in the amidohydrolase superfamily of metalloenzymes, but previous studies suggested that a metal was not required for activity. To help resolve that conundrum, a double chaperone expression system was used to produce multimilligram quantities of functionally folded, recombinant TrzN. The TrzN obtained from Escherichia coli (trzN) cells cultured with increasing zinc in the growth medium showed corresponding increases in specific activity, and enzyme obtained from cells grown with 500 muM zinc showed maximum activity. Recombinant TrzN contained 1 mole of Zn per mole of TrzN subunit. Maximally active TrzN was not affected by supplementation with most metals nor by EDTA, consistent with previous observations (E. Topp, W. M. Mulbry, H. Zhu, S. M. Nour, and D. Cuppels, Appl. Environ. Microbiol. 66:3134-3141, 2000) which had led to the conclusion that TrzN is not a metalloenzyme. Fully active native TrzN showed a loss of greater than 90% of enzyme activity and bound zinc when treated with the metal chelator 8-hydroxyquinoline-5-sulfonic acid. While exogenously added zinc or cobalt restored activity to metal-depleted TrzN, cobalt supported lower activity than did zinc. Iron, manganese, nickel, and copper did not support TrzN activity. Both Zn- and Co-TrzN showed different relative activities with different s-triazine substrates. Co-TrzN showed a visible absorption spectrum characteristic of other members of the amidohydrolase superfamily replaced with cobalt.
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PMID:TrzN from Arthrobacter aurescens TC1 Is a zinc amidohydrolase. 1688 54

Alpha-amino-beta-carboxymuconate-epsilon-semialdehyde decarboxylase (ACMSD) is a widespread enzyme found in many bacterial species and all currently sequenced eukaryotic organisms. It occupies a key position at the branching point of two metabolic pathways: the tryptophan to quinolinate pathway and the bacterial 2-nitrobenzoic acid degradation pathway. The activity of ACMSD determines whether the metabolites in both pathways are converted to quinolinic acid for NAD biosynthesis or to acetyl-CoA for the citric acid cycle. Here we report the first high-resolution crystal structure of ACMSD from Pseudomonas fluorescens which validates our previous predictions that this enzyme is a member of the metal-dependent amidohydrolase superfamily of the (beta/alpha)(8) TIM barrel fold. The structure of the enzyme in its native form, determined at 1.65 A resolution, reveals the precise spatial arrangement of the active site metal center and identifies a potential substrate-binding pocket. The identity of the native active site metal was determined to be Zn. Also determined was the structure of the enzyme complexed with cobalt at 2.50 A resolution. The hydrogen bonding network around the metal center suggests that Arg51 and His228 may play important roles in catalysis. The metal center configuration of PfACMSD is very similar to that of Zn-dependent adenosine deaminase and Fe-dependent cytosine deaminase, suggesting that ACMSD may share certain similarities in its catalytic mechanism with these enzymes. These data enable us to propose possible catalytic mechanisms for ACMSD which appear to be unprecedented among all currently characterized decarboxylases.
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PMID:Crystal structure of alpha-amino-beta-carboxymuconate-epsilon-semialdehyde decarboxylase: insight into the active site and catalytic mechanism of a novel decarboxylation reaction. 1693 94

The majority of soil microorganisms can derive ethylene from L-methionine (L-MET), while some rhizobacteria can hydrolyze 1-aminocyclopropane-1-carboxylate (ACC) due to their ACC-deaminase activity. In this study, three strains having either ACC-deaminase activity (Pseudomonas putida biotype A, A7), or the ability to produce ethylene from L-MET (Acinetobacter calcoaceticus, M9) or both (Pseudomonas fluorescens, AM3) were used for inoculation. The highly ethylene specific bioassay of a classical "triple" response in pea seedlings was used to investigate the effect of the inoculation with the rhizobacteria in the presence of 10 mM ACC or L-MET. The exogenous application of ACC had a concentration-dependent effect on the etiolated pea seedlings in creating the classical "triple" response. The inoculation with P. putida diluted the effect of ACC, which was most likely due to its ACC-deaminase activity. Similarly, the application of Co2+ reduced the ACC-imposed effect on etiolated pea seedlings. In contrast, the inoculation of A. calcoaceticus or P. fluorescens in the presence of L-MET caused a stronger classical "triple" response in etiolated pea seedlings; most likely by producing ethylene from L-MET. This is the first study, to our knowledge, reporting on the comparative effect of rhizobacteria capable of utilizing ACC vs L-MET on etiolated pea seedlings.
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PMID:Differential response of etiolated pea seedlings to inoculation with rhizobacteria capable of utilizing 1-aminocyclopropane-1-carboxylate or L-methionine. 1734 50

NagA is a member of the amidohydrolase superfamily and catalyzes the deacetylation of N-acetyl-d-glucosamine-6-phosphate. The catalytic mechanism of this enzyme was addressed by the characterization of the catalytic properties of metal-substituted derivatives of NagA from Escherichia coli with a variety of substrate analogues. The reaction mechanism is of interest since NagA from bacterial sources is found with either one or two divalent metal ions in the active site. This observation indicates that there has been a divergence in the evolution of NagA and suggests that there are fundamental differences in the mechanistic details for substrate activation and hydrolysis. NagA from E. coli was inactivated by the removal of the zinc bound to the active site and the apoenzyme reactivated upon incubation with 1 equiv of Zn2+, Cd2+, Co2+, Mn2+, Ni2+, or Fe2+. In the proposed catalytic mechanism the reaction is initiated by the polarization of the carbonyl group of the substrate via a direct interaction with the divalent metal ion and His-143. The invariant aspartate (Asp-273) found at the end of beta-strand 8 in all members of the amidohydrolase superfamily abstracts a proton from the metal-bound water molecule (or hydroxide) to promote the hydrolytic attack on the carbonyl group of the substrate. A tetrahedral intermediate is formed and then collapses with cleavage of the C-N bond after proton transfer to the leaving group amine by Asp-273. The lack of a solvent isotope effect by D2O and the absence of any changes to the kinetic constants with increases in solvent viscosity indicate that net product formation is not limited to any significant extent by proton-transfer steps or the release of products. N-Trifluoroacetyl-d-glucosamine-6-phosphate is hydrolyzed by NagA 26-fold faster than the corresponding N-acetyl derivative. This result is consistent with the formation or collapse of the tetrahedral intermediate as the rate limiting step in the catalytic mechanism of NagA.
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PMID:N-Acetyl-D-glucosamine-6-phosphate deacetylase: substrate activation via a single divalent metal ion. 1756 47

The microbial degradation of nitriles is of interest for bioremediation and green chemistry. We demonstrated that the soil bacterium Rhodococcus sp. RHA1 utilizes a range of nitriles, including acetonitrile, as growth substrates. Proteomic analysis identified 13 proteins that were more abundant in acetonitrile-grown cells, including an aliphatic amidase and a protein with no known homologue. Purification of a nitrile hydratase (NHase) from acetonitrile-grown cells identified the unknown protein as the beta subunit of a two-subunit NHase. Sequence analysis revealed that the genes encoding the amidase (anhC) and the NHase (anhAB) occur in a 12.8 kbp cluster located on plasmid pRHL2. The anh gene cluster also encodes an acetyl-CoA hydrolase, transcriptional regulators, a putative cobalt transporter and a protein of unknown function. Striking features of the NHase include the amino acid sequence identity (32%) and large size (63 and 56 kDa) of the alpha and beta subunits, as well as the enzyme's metal ion content (one cobalt, two copper and one zinc). The enzyme possessed similar specificities for acetonitrile and propionitrile (k(cat)/K(m) approximately 7 mM(-1) s(-1)) followed by acrylonitrile and butyronitrile. We propose that this acetonitrile hydratase (ANHase) represents the first member of a previously unknown class of NHases.
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PMID:Purification and characterization of a novel nitrile hydratase from Rhodococcus sp. RHA1. 1763 93


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